《环境昆虫学报》

【目的】短头熊蜂Bombus breviceps是我国西南地区优势熊蜂种，其分布广，群势大，存在体色模式多且复杂的现象，本研究旨在分析短头熊蜂体色多态性形成过程黑色素合成的相关基因情况。【方法】通过PCR技术扩增短头熊蜂的黑色素合成相关基因ebony（N-β-丙酰生物胺合成酶）CDS区，并进行生物信息学和系统进化分析；同时，利用荧光定量PCR检测该基因在短头熊蜂不同发育阶段（卵、幼虫、蛹、成虫）及1日龄成年工蜂不同组织（头、胸、腹、肠道、蛰针、足、翅膀）的表达水平。【结果】结果显示短头熊蜂ebony基因（简称Brebony）CDS区全长2 583 bp，编码860个氨基酸（GenBank登录号：PV007825），预测分子量97.02 kDa，理论等电点5.10，具有典型的非核糖体肽合成酶结构域。系统进化分析显示短头熊蜂Ebony蛋白与熊蜂属的昆虫聚为一支，与膜翅目无刺蜂属和蜜蜂属亲缘较近。时空表达谱显示，相比于1日龄卵、整个幼虫期、预蛹、蛹期（1，3，5，7，13，15日龄蛹）以及1日龄成蜂，Brebony基因在3日龄和5日龄卵、蛹期（9，11日龄蛹）以及刚羽化成蜂上有较高表达；在1日龄成年工蜂各组织中，翅膀表达量最高，其次是足、胸、腹、头、蛰针，在肠道上表达量最低。【结论】综上，短头熊蜂Brebony基因编码的生物胺合成酶的蛋白序在熊蜂属内保守性高，参与短头熊蜂工蜂羽化色素沉积及卵的胚胎发育、孵化等过程，可为解析短头熊蜂体色多态性形成的分子机制提供参考。

英文摘要:

【Aim】Bombus breviceps is a dominant species of bumblebee in the southwestern China. It exhibits a wide distribution, large population size, and displays multiple and complex color patterns. In this study, the gene associated with melanin synthesis during the formation of its body color polymorphism was analyzed. 【Methods】The coding sequence of the ebony gene (encoding Ebony protein, an N-β-alanyl biogenic amine synthetase) was amplified by PCR. Subsequently, bioinformatics and phylogenetic analyses were conducted. Additionally, quantitative PCR was performed to assess the expression level of ebony during different developmental stages (egg, larva, pupa and adult) as well as in various tissues (head, thorax, abdomen, gut, sting, legs and wings) of 1-day-old workers. 【Results】The results showed that the coding sequence of the ebony gene in B. breviceps (abbreviated as Brebony) was 2 583 bp and encoded 860 amino acids (GenBank accession number: PV007825). The predicted molecular weight was 97.02 kDa with a theoretical isoelectric point of 5.10. Furthermore, it possessed a typical nonribosomal peptide synthase domain. Phylogenetic analysis showed that Ebony protein of B.breviceps was clustered with the genus Bombus and closely related to those from Trigona and Apis species in the Hymenoptera. Spatio-temporal expression profiles showed that, compared with 1-day-old eggs, larval stage, pupal stage (1-, 3-, 5-, 7-, 13- and 15-day-old pupae) and – workers, Brebony had relatively higher expression levels in the 3-day-old and 5-day-old eggs, the pupal stages (9- and 11-day-old pupae), and in newly emerged adults. Among the tissues of 1-day-old workers, the expression level was the highest in wings, followed by expression levels in legs, thorax, abdomen, head and sting. The lowest expression level was detected in the gut. 【Conclusion】In conclusion, the protein sequence of the biogenic amine synthetase encoded by Brebony exhibits a relatively high degree of conservation in Bombus. Moreover, it is involved in the processes such as pigment deposition during worker eclosion, embryonic development and hatching in B. breviceps. This study provides a insight into analyzing the molecular mechanisms underlying the formation of color-pattern polymorphism in B. breviceps.

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